System and method for enhancing confocal reflectance images of tissue specimens

ABSTRACT

A confocal scanning microscope system ( 10 ) using cross polarization effects and an enhancement agent (acetic acid) to enhance confocal microscope reflectance images of the nuclei of BCCs (basal cell carcinomas) and SCCs (squamous call carcinomas) in the confocal reflectance images of excised tumor slices. The confocal scanning microscope system having a laser ( 11 ) for generating an illumination beam ( 12 ), a polygon mirror ( 18 ) for scanning the beam to a tissue sample ( 22 ) and for receiving a return beam from the tissue sample and detector ( 28 ) for detecting the returned beam to form an image. The system further includes a half-waveplate ( 13 ) having a rotatable stage ( 14 ) and a quarter-wave plate ( 21 ) having a rotatable stage ( 20 ) disposed in the optical path of the illumination beam and at least a linear polarizer ( 24 ) having a rotatable stage ( 25 ) disposed in the optical path of the returned beam from the tissue sample.

This application claims the benefit of priority to U.S. ProvisionalApplication Nos. 60/125,033, filed Mar. 18, 1999, and 60/146,819, filedAug. 2, 1999, which are herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to confocal microscopy and particularly toa system (method and apparatus) for enhancing images of tissue at thesurface or internally of a tissue sample so as to enable rapid andaccurate screening of tissue for the determination of the nuclear andcellular structure thereof The present invention also relates to amethod for diagnosing cancerous cells in skin tissue using confocalmicroscopy. The invention is especially suitable in providing enhancedimages of the nuclei of BCC/SCC (basal cell carcinoma or squamous cellcarcinoma) in confocal reflectance images of tumor slices obtainedduring Mohs micrographic surgery. Tissue may be either naturallyexposed, or surgically excised tissue.

BACKGROUND OF THE INVENTION

Mohs micrographic surgery for BCCs and SCCs involves precise excision ofthe cancer with minimal damage to the surrounding normal skin.Conventionally, precise excision is guided by histopathologicexamination for cancer margins in the excised tissue slices during Mohssurgery. Typically, 2-4 slices are excised, and there is a waiting timeof 10-30 minutes for the surgeon and patient while each slice is beingprocessed.

Confocal reflectance microscopes can noninvasively image nuclear andcellular detail in living skin to provide images of tissue sections,such a microscope is described in U.S. Pat. No. 5,880,880. The contrastin the images is believed to be due to the detected variations in thesingly back-scattered light caused by variations in refractive indicesof tissue microstructure. Within the epidermal (basal and squamous)cells, the cytoplasm appears bright and the nuclei as dark ovals. Theunderlying dermis consists of collagen bundles and that, too, appearsbright with dark spaces in-between. Thus, when neoplasticepidermal-cells invade the dermis as in BCCs and SCCs, confocaldetection of the cancers is very difficult because the cells and nucleilack contrast relative to the surrounding normal dermis.

SUMMARY OF THE INVENTION

It is the feature of the present invention to provide an improved systemand method for confocal microscopy by cross polarizing the lightilluminating a tissue sample and the light returned from the tissuesample representing a section of the tissue.

It is another feature of the present invention to use such crosspolarizing of the light illuminating a tissue sample and the lightreturned from the tissue sample in combination with imaging the samplewhen immersed in an image enhancement agent.

It is a further feature of the present invention to provide a method fordiagnosing cancerous cells in skin tissue using confocal microscopy

Briefly described, a system for providing enhanced images in confocalmicroscopy is provided by utilizing cross polarized light in theillumination of tissue and in the detection of light from which theimages are formed, respectively, and where an image enhancing agent,such as acetic acid or vinegar solution, is used in a bath in which thespecimen is immersed while being imaged.

It has been found in accordance with the invention that a confocal laserscanning microscope using cross polarized components of light inillumination and in the detection of the reflected light from tissuespecimens immersed in such an enhancement agent solution images of thecellular structure are enhanced, enabling cells and voids in thestructure and the cell condition to be readily observed. By virtue ofthe use of such cross polarized light in imaging of tumor slicesobtained in the course of Mohs surgery, epidermis sections which mayhave holes in the collagen are imaged more accurately so that holes areunlikely to be confused with cells or cell structure.

A method is also provided for detecting cancerous basal cell andsquamous cell in dermal tissue with confocal reflected light imaginghaving the steps of washing the tissue to be imaged with a solution ofacetic acid to whiten epithelial cells and compact chromatin of thetissue; imaging the tissue with a confocal microscope to provideconfocal images of basal and squamous cells in which the confocalmicroscope directs light into the tissue and collects reflected lightrepresenting confocal images of the tissue; changing the polarizationstate of the light used by the confocal microscope to increase thecontrast of the nuclei of basal and squamous cells in the confocalimages; and analyzing the nuclei of the basal and squamous cells in theconfocal images to diagnose which of such cells are cancerous.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing features and advantages of the invention will become moreapparent from a reading of the following description in connection withthe accompanying drawings wherein,

FIG. 1 is a schematic diagram of a Vivascope (TM) confocal microscopewhich is available from Lucid Inc. of Rochester, N.Y. and is describedin the above referenced U.S. Pat. No. 5,880,880; and

FIGS. 2A, B, C, and D are schematic depictions of various parts of theconfocal microscope system and the cross-polarized illumination which isused therein.

DETAILED DESCRIPTION OF INVENTION

Referring to the drawings, in the confocal microscope 10 of FIG. 1, alinearly polarized (p-state) laser beam 12 is passed through a half waveplate (HWP) 13 on a rotation stage 14. A confocal microscope especiallysuitable in practicing the invention is described in U.S. Pat. No.5,880,880, issued Mar. 9, 1999, which is herein incorporated byreference. Other confocal microscopes may also be used. The illuminationthrough the non-polarizing or partially polarizing beam splitter 16 isscanned, as by a polygon mirror 18 and galvanometric mirror 19 acrossthe specimen or sample 22 having a surface 22 a. As shown in FIGS. 2Band 2C, sample 22 may be a BCC/SCC sample in a sample holder orcontainer 22 b contained in an enhancement solution bath 26 having water28 under a tissue ring 33 which places the sample 22 under tension. Asshown in FIG. 1, the microscope 10, via an objective lens 23, images thetissue sample 23 through an opening 33 a in the tissue ring 33. Forexample, the opening 33 a may include a window having a materialtransparent to the beam.

The target surface is the surface of the sample 22 (such as a tissuetumor specimen), which may be at the surface 22 a or within the body ofthe sample, utilizing the techniques described in the above referencedU.S. Patent. The polarization of the incident light and the reflectedlight also can be modified using a quarter wavelength plate (QWP) 21which is also removably mounted on a rotation stage 20.

The detected light is cross-polarized that is in the s-state as shown bythe bulls-eye indication 12 a in FIG. 1 and labeled “detection s-state”in FIG. 2D. It is crossed or perpendicular or orthogonal to the p-state.Although preferably cross-polarized light is in s and p states, becausethe beam splitter may be non-polarizing or partially polarizing, otherstates are possible. The detected illumination of desired polarizationis obtained with an analyzer 24 also mounted on a rotation stage 25. Forexample, analyzer 24 may be a linear polarizer. The light from theanalyzer 24 is passed through the confocal aperture 28 a, such as apinhole, and a photo-detector 28, such as an avalanche photodiode (APD)in FIG. 1. While p polarized light from a linearly polarized laser 11 isshown in FIG. 1, the linearly polarized laser 11 and the half wave plate13 can be replaced with a laser providing an unpolarized laser beam anda linear polarizer, respectively. Further, the linear polarizer and theanalyzer 24 can then be replaced with a polarized beam splitter. Also,instead of rotating the half wave plate 13 and the analyzer 24, they canbe kept fixed in cross polarization states and the sample 22 can berotated.

As shown in FIG. 1, optical components are provided in confocalmicroscope 10 to direct the beam from laser 11 along a path to sample22, and include, beam expander-spatial filter 42 (which, for example,may be provided by two lens 42 a and 42 b and aperture 42 c), HWP 13,mirror 43, ND filter 44 (which, for example, may be a neutral densityfilter, such as provided by a circular variable attenuator manufacturedby Newport Research Corporation), through beam splitter 16 to polygonmirror 18. The beam is then deflected by polygon mirror 18 through alens 45 (which for example, may be a f/2 lens), a lens 46 (which forexample, may be a f/5.3 lens), and deflected by galvanometric mirror 19through a lens 47 (which for example, may be a f/3 lens), QWP 21 andobjective lens 23 to sample 22. The optical components along the path ofthe reflected light returned from the sample 22 to detector 28 include,objective lens 23, QWP 21, lens 47, and deflected by mirrors 19 and 18via lenses 46 and 45 to beam splitter 16. The beam splitter 16 directsthe returned light through lens 48, analyzer 24, and pinhole 28 a todetector 28. The raster line 17 a and raster plane 17 b in FIG. 1 areillustrated by dashed lines to denote the angular scan of the beam alonga raster line 17 agenerated by the rotation of polygon mirror 18, whilethe angular movement of galvanometric mirror 19 scans that raster lineto form a raster plane 17 b. In this manner, a confocal image of atissue section can be captured by the control electronics 38 throughdetector 28. To provide a start of scan beam 12 c to synchronize thecontrol electronics 38 with the start of each raster line, the beamsplitter 16 directs part of the beam incident the beam splitter 16 torotating polygon mirror 18, via mirror 48, to split diode 50 (e.g.,photo-diode) which is connected to the control electronics 38 to providea start of scan pulse at the beginning of each raster line. Motors, notshown, can provide the desired rotation and angular movement ofrespective mirrors 18 and 19.

The system which is shown in FIG. 1 operates as follows:

1. Remove QWP 21. Rotate the HWP 13 so that its fast axis is at 90degrees with the illumination p-state (see FIG. 2A). Thus, there is nochange (rotation) of the direction of the p-state. Rotate the analyzer24 so that it acts as a crossed polarizer and transmits the detections-state (which is orthogonal to the illumination p-state).

2. The surgically excised tissue sample 22 is placed in a water bath 26with a tissue-ring 33 placed on top (see FIG. 2B).

3. The water bath 26 containing the sample 22 is placed under theobjective lens 23, such that the tissue-ring 33 fits into the objectivelens housing 31 (see FIG. 2C). The water bath 26 is on an XY translationstage 34 to move the sample 22. The XY stage 34 is on a lab-jack 35 withwhich can move the entire assembly 36 upwards, such that the sample 22is gently pressed between the tissue-ring 33 and the water bath 26 tokeep the sample 22 still during the imaging. Arrow 37 denotes thedirection of such light pressure.

4. Rotate the HWP 13 in small angular increments of 10 degrees and,correspondingly, the analyzer 24 in angular increments of 20 degrees, ontheir respective stages 14 and 25, such that the analyzer 24 is alwayscross-polarized with respect to the illumination polarization state. Theconfocal images of the sample 22 change from bright to dark to bright asthe HWP 13 and analyzer 24 is rotated.

5. Set the HWP 13 and analyzer 24 such that the sample 22 appears dark(i.e., minimum brightness). Survey the sample 22 by moving it with theXY stage 34, to check that the sample appears dark everywhere in theconfocal images.

6. Lower the water bath 26 using the lab-jack 35. Remove the water fromwithin the tissue ring 33, and add an enhancement agent, namely aceticacid (e.g., to provide a 5% by volume—ph 2.5—solution in the water).Raise the lab-jack 35 and place the sample 22, as before, under theobjective lens 23.

7. Survey the sample 22 by moving it with the XY stage 34, and focusingon the surface and at varying depths of the sample with the objectivelens 23 (which may be mounted on a Z-translation stage to move theobjective lens towards and away from the sample). Confocal images areeither videotaped or grabbed in this “crossed polarization” mode at aframe grabber 39, video monitor 40, or videotape recorder 41 via controlelectronics 38.

8. Whenever or wherever necessary, confocal images are obtained in“brightfield” mode, to either determine lateral or depth location, oridentify structures (examples: hair follicles, sweat ducts, epithelialmargins) within the sample. (This is analogous to using reflectanceimaging in conjunction with fluorescence imaging.) The QWP 21 isinserted and rotated so that its optic axis is at 45 degrees to both theillumination and detection linear polarization states (see FIG. 2D).

With the confocal reflectance light microscope 10 described herein,BCCs, SCCs in human skin are described herein without the processing(fixing, sectioning, staining) that is required for conventionalhistopathology of Mohs surgery. Rapid confocal detection is providedafter strongly enhancing the contrast of nuclei in the cancer cellsrelative to the surrounding normal tissue using acetic acid and crossedpolarization.

To improve the detection of BCCs and SCCs in confocal images in tissue,such as dermal tissue, which may be either naturally exposed, orsurgically excised, the contrast of the nuclei of such cells isincreased by the following method. The area of the tissue to be imagedis washed with 5% acetic acid, as described earlier. Acetic acid causeswhitening of epithelial tissue and compaction of chromatin. Thechromatin-compaction is believed to increase its refractive index, whichthen increases light back-scatter from the nuclei and makes them appearbright. Next, the tissue area is imaged with confocal microscope 10 inwhich the polarization state of the light directed to the tissue andcollected by the confocal microscope is controlled by rotating thelinear polarizer of analyzer 24. When illuminated with linearlypolarized light and confocally imaged through the analyzer 24, thebrightness of the acetic acid-stained nuclei does not vary much, whereasthe brightness of the collagen varies from maximum to minimum. Theback-scattered light from the inter-nuclear structure is significantlydepolarized (probably due to multiple scattering), whereas that from thedermis preserves the illumination polarization (due to singleback-scatter). With the light in a crossed polarized state, brightnuclei in the BCCs and SCCs are shown in the confocal images produced bythe microscope in strong contrast against a dark background ofsurrounding normal dermis. BCCs and SCCs can be distinguished fromnormal tissue by the cellular organization, cell size, cell shape,nuclear morphology, and cellular differentiation. One example ofcellular organization is anaplasia. One example of cell size and shapeand nuclear morphology is dysplasia. One example of cellulardifferentiation is pleomorphism.

Thus, the bright clusters of nuclei in the cancer cells are detectableat low resolution, as in conventional histopathology. Mosaics oflow-resolution confocal images can be assembled to produce confocal mapsof the BCCs or SCCs within the entire excised tissue. Detection of thecancers is made within minutes; thus, the total savings in time for aMohs surgery can be hours.

Others cancers and tissue abnormalities may also be detected by usingthis approach any time a cellular tissue needs to be distinguished froma cellular background. For example, dermal melanocytes, mucosal tissuein stromal tissue, breast epithelium in a stromal matrix.

From the foregoing description, it will be apparent that an improvedsystem for enhanced imaging in confocal microscopy and method fordiagnosing skin cancer cells have been described. Variations andmodifications in the herein described system, method, and in theenhancement agent used therein will undoubtedly become apparent to thoseskilled in the art. Accordingly, the foregoing description should betaken as illustrative and not in a limiting sense.

What is claimed is:
 1. A system for confocal imaging tissue comprising:means for generating an illumination beam; optics for scanning the beamto the tissue and receiving returned illumination from the tissuerepresenting a section of the tissue in which said optics has means forcontrolling the polarization state of the illumination beam and thereturned illumination to enable cross-polarization of the illuminationbeam with respect to the returned illumination; and means for detectingthe returned illumination to form an image of the section of the tissue.2. The system according to claim 1 wherein said polarization statecontrolling means comprises means for maintaining saidcross-polarization of the illumination beam and the returnedillumination from the tissue when the polarization state of theillumination beam and returned illumination are changed.
 3. The systemaccording to claim 2 wherein said light in the illumination beam ispolarized in a p-state and the returned illumination is polarized to ans-state.
 4. The system according to claim 1 wherein said sample islocated in a solution which enhances the brightness of one or moretissue structures in the image of the section of the tissue.
 5. Thesystem according to claim 4 wherein said solution has an acid component.6. The system according to claim 5 wherein said acid component is one ofacetic acid and vinegar.
 7. The system according to claim 1 whereintissue represent the excised tissue of a patient, said optics compriseat least an objective lens for focusing the illumination beam to thetissue and collecting returned illumination from the tissue, and saidsystem further comprises: a container having a liquid in which saidtissue is disposed in said container in said liquid; and means forplacing said tissue under tension against a surface in said containerwhile enabling imaging of the tissue by said objective lens.
 8. Thesystem according to claim 1 further comprising means for moving saidsample with respect to said objective lens.
 9. The system according toclaim 1 wherein said means for controlling the polarization statecomprises means capable of changing the polarization of the illuminationbeam and the polarization of the returned illumination from the sample.10. The system according to claim 9 wherein said means capable ofchanging the polarization comprises a half-wave plate and a quarter-waveplate through which passes said illumination beam to said sample, and alinear polarizer through which passes the returned illumination fromsaid sample.
 11. The system according to claim 10 wherein at least oneof said half-wave plate, quarter-wave plate, and linear polarizer havemeans for rotation to change the polarization of the light passing therethrough.
 12. The system according to claim 1 wherein said tissue has animage enhancing agent, and said polarization controlling means iscapable changing the polarization state of at least one of theillumination beam and the returned illumination to effectcharacteristics of tissue structures in the image of the tissue sectionto enable determination of which of the tissue structures are cancerous.13. The system according to claim 12 wherein said tissue is skin tissue.14. The system according to claim 1 wherein the tissue is one ofnaturally exposed tissue and surgically excised tissue.
 15. A system forproviding enhanced images in confocal microscopy which is comprisingmeans for utilizing cross polarized light in the illumination of tissueand in the detection of light from which the images are formed,respectively, and wherein an image enhancing agent is used in a bath inwhich the specimen is immersed while being imaged.
 16. The systemaccording to claim 15 wherein said image enhancing agent is one ofacetic acid and vinegar.
 17. A method for confocal imaging tissuecomprising the steps of: generating an illumination beam; scanning thebeam to the tissue; receiving returned light from the tissuerepresenting of a section of the tissue; cross polarizing theillumination beam and the returned light with respect to each other; anddetecting the returned light to form an image of the section of thetissue.
 18. The method according to claim 17 further comprising the stepof locating said sample in a solution which enhances the brightness ofone or more tissue structures in the image of the section of the tissue.19. The method according to claim 18 wherein said solution has an acidcomponent.
 20. The method according to claim 19 wherein said acidcomponent is one of acetic acid and vinegar.
 21. The method according toclaim 17 wherein said tissue has an image enhancing agent, and saidpolarization controlling step further comprises the step of changingpolarization state of at least one of the illumination beam and thereturned light to effect characteristics of tissue structures in theimage of the tissue section to enable determination of which of thetissue structures are cancerous.
 22. The method according to claim 21wherein said tissue is skin tissue.
 23. The system according to claim 17wherein the tissue is one of naturally exposed tissue and surgicallyexcised tissue.
 24. A method for detecting cancerous basal cell andsquamous cell in dermal tissue with confocal reflected light imaging,said method comprising the steps of: washing the tissue to be imagedwith a solution which whitens epithelial cells and compacts chromatin ofthe tissue; imaging the tissue with a confocal microscope to provideconfocal images of basal and squamous cells in which the confocalmicroscope directs light into the tissue and collects reflected lightrepresenting confocal images of the tissue; changing the polarizationstate of the light used by the confocal microscope to increase thecontrast of the nuclei of basal and squamous cells in the confocalimages; and analyzing the nuclei of the basal and squamous cells in theconfocal images to diagnose which of such cells are cancerous.
 25. Themethod according to claim 24 wherein said solution is of acetic acid.26. A system for confocal imaging tissue comprising: means forgenerating an illumination beam; optics for scanning the beam to thetissue and receiving returned illumination from the tissue representinga section of the tissue in which said optics has means for controllingthe polarization state of the illumination beam and the returnedillumination; means for detecting the returned illumination to form animage of the section of the tissue, and a solution in which said tissueis located capable of enhancing the brightness of one or more tissuestructures in the image of the section of the tissue.
 27. The systemaccording to claim 26 wherein said solution enhances the brightness ofone or more tissue structures in accordance with the polarization stateof the illumination beam and the returned illumination.
 28. The systemaccording to claim 26 wherein said solution has an acid component. 29.The system according to claim 28 wherein said acid component is one ofacetic acid and vinegar.